Methods for identifying an analog that promotes nerve regeneration

Abstract

Analogs of FK506 that do not bind FKBP-12 have been found to effectively promote nerve cell growth and regeneration, thereby speeding functional recovery of damaged nervous tissue and axonal regeneration without causing immunosuppression.

1. A method of identifying a FK506 analog that stimulates nerve cell growth, the method comprising:
 screening a plurality of FK506 analogs for binding to FKBP-12 and for rotamase activity; and 
 selecting a FK506 analog of interest that binds FKBP-12 with a K d  of at least 10 μM without inhibition of FKBP-12 rotamase activity; 
 thereby identifying a FK506 analog that stimulates nerve growth. 
 
     
     
       2. the method of  claim 1 , further comprising assaying the FK506 analog for activity in promoting nerve cell growth. 
     
     
       3. The method of  claim 2 , wherein the assay for activity in promoting nerve cell growth comprises exposing a cell to the selected FK506 analog and determining if neurite outgrowth is promoted. 
     
     
       4. The method of  claim 3 , wherein the cell is a neuroblastoma cell. 
     
     
       5. The method of  claim 3 , wherein the cell is a sciatic nerve cell. 
     
     
       6. The method of  claim 1 , wherein the FK506 analog binds FKBP-12 with a K d  of at least 30 μM. 
     
     
       7. The method of  claim 1 , wherein the FK506 analog binds FKBP-12 with a K d  of at least 100 μM. 
     
     
       8. A method of identifying a FK506 analog that stimulates nerve cell growth, the method comprising:
 screening FK506 analogs for binding to FKBP-12 in vitro; 
 selecting one or more FK506 analogs of interest that bind FKBP-12 with a K d  of at least 10 1 μM without inhibition of rotamase activity, wherein the one or more FK506 analogs of interest do not inhibit FKB-12 rotamase activity in vivo when administered to a patient at a dosage level up to about 100 mg/kg body weight/day; and 
 determining if the one or more FK506 analogs of interest promotes nerve cell growth in an assay for nerve cell growth, wherein the assay for nerve cell growth is an in vivo or an in vitro assay. 
 
     
     
       9. The method of  claim 8 , wherein the assay for nerve cell growth comprises exposing a cell to the one or more FK506 analogs of interest in vitro and determining if neurite outgrowth is promoted. 
     
     
       10. The method of  claim 9 , wherein the cell is a neuroblastoma cell. 
     
     
       11. The method of  claim 9 , wherein the FK506 analog binds FKBP-12 with a K d  of at least 30 μM. 
     
     
       12. The method of  claim 9 , wherein the FK506 analog binds FKBP-12 with a K d  of at least 100 μM. 
     
     
       13. The method of  claim 8 , wherein the assay for nerve cell growth comprises exposing a sciatic nerve cell to the one or more FK506 analogs of interest. 
     
     
       14. The method of  claim 8 , wherein the assay for nerve cell growth is an in vivo assay.